The preparation of a highly specific substrate for a given proteolytic enzyme usually requires the synthesis of specific oligopeptide of sufficient length to interact with as many subsites of the particular active site as possible. The hydrolysis of such an oligopeptide can be followed by a number of methods such as colorimetry, potentiometry, or spectroscopy. The latter method is usually preferred because of high sensitivity and convenience.
Spectroscopic monitoring of proteolytic activity is possible only if a spectral change occurs during the cleavage of a specific bond linking a suitable chromophore to the rest of the molecule.